Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame

Biochimica et Biophysica Acta, 1182(1993) 157-161

157

© 1993 Elsevier Science Publishers B.V. All rights reserved 0925-4439/93/$06.00

BBADIS 61292

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame Dalibor Stys a,1, Ivo Blaha

a

and Peter Strop a,2

a Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague (Czechoslocakia)

(Received 11 February 1993)

Key words: HIV-1; gag; p6; Protein structure; Proteinase; Open reading frame Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

Introduction Protein p6 is the last protein encoded by the gag open reading frame of HIV-1 virus. Not all retroviruses encode this protein. P6 and its analogues are characteristic of the retroviruses that have a cone-shaped core. Its high content of Pro and hydrophilic residues is typical for this group of proteins. Little is known about the function of p6. Gottlinger et al. [1,2] found that the p6-deficient HIV-1 virions are non-infective. They form large number of buds but new virions are not released from the cell surface. The p6 protein probably plays a role in the final assembly of the viral particle. Gelderblom [3] proposed the hy-

Correspondence to: Dalibor Stys, Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Flemingovo n. 2, 16610 Praha, Czechoslovakia. i Present address: Plant Cell Biology, Lund University, Box 7007, S-22007 Lund, Sweden. e Present address: Selectide Corporation, 10900 New Stallard PI., Suite 122, Tucson, AZ 85737, USA. Abbreviations: HIV, human immunodefficiencyvirus; Boc, butyoxycarbonile; PAM, phenylacetamidomethyl; Tos, toluensulfonile; OcHex, cyclohexylester;oCIZ, orthochlorbenzoylcarbonile;Bzl, benzyl; For, formyl; oBzl, orthobenzyl.

pothesis that p6 is present in the core-envelope link. It is consistent with on the observation of Roberts and Oroszlan [4], who found an analogous protein in E I A V virus in the virion but not in the core. Zybarth et al. [5] have found that processing of p6 deficient gag protein by HIV-1 proteinase is faster than that of the wild type. They proposed the hypothesis that p6 is involved in initiation of processing. The p6 protein is expected to form a region which limits the interaction of the proteinase with the rest of the polyprotein to be processed. According to this hypothesis, release of p6 should thus be the activation step in the polyprotein maturation. We think that both the observation of Gottlinger et al. [1,2] and tthat of Zybarth et al. [5] can eventually be explained if the p6 protein acts as a strong inhibitor of HIV-1 proteinase. P6 would then slow down the rate of gag maturation and prevent further cleavage of the mature structural proteins by HIV-1 proteinase in the late stage of maturation.

Materials and Methods Structure prediction

The secondary structure of protein p6 was predicted by the programs of Chou and Fasman [6,7] and Garnier

158 et al. [8] using the DNASIS program package (LKB). The consensus sequence used for prediction was build from the amino acid sequences which occur most frequently at certain position in various isolates. The sequences for the isolates were taken from EMBL data bank of DNA sequences [9].

Protein synthesis The p6 protein of the sequence found in the isolate Bru [9] was synthesised by Boc strategy of peptide synthesis [10] on Boc-GIy-PAM resin (0.5 mmol/g). The protected amino acids were used as follows Arg(Tos), Asp(OcHex), Lys(OC1Z), Ser(Bzl), Thr(Bzl), Trp(For), Tyr(oBzl). The crude product was then purified by reverse-phase HPLC, using Vydac C~a protein column and linear H20-acetonitrile gradient. The purified fractions were analysed by amino acid analysis. The amino acid composition of the largest fraction was in good agreement with the expected one. Protein refolding A solution of p6 protein at 0.8 mM concentration was dialysed for 12 h against 0.5 M phosphate buffer (pH 5.7) containing 8 M urea. The solution was then stepwise dialysed against buffers containing 6, 4, 2 and 0 M urea for 4 h each in the first experiment and for 8 h in the second experiment. The same experiments were performed at pH 6.5, 6 and 5.5 in 0.5 M phosphate buffer and at pH 4.5, 4 and 3.5 in 0.1 M acetate buffer. In the temperature refolding experiment, the p6 protein solution in 0.5 M phosphate buffer (pH 5.4) was heated to 65°C and then slowly cooled to 25°C in 3 h in the first experiment and in 12 h in the second experiment. CD spectra in the near-UV region CD spectra were measured in the range from 190 to 260 nm using a Jobin-Yvon Dichrograph Mark V. The concentration of the protein was 0.05 mM in 0.05 M phosphate buffer (pH 5.4). Cell pathlengths of 1 mm and 1 cm were used for far- and near-UV ranges, respectively. Spectra were recorded in 8 scans at 0.5 nm intervals with a time constant of 2 s. The spectra were analysed for the content of secondary structure elements by program developed in house according to the standard spectra data bases published by Chou and Fasman [11] and Bolotina et al. [12]. One-dimensional NMR spectra The proton NMR spectra were acquired in 16 scans using a Varian UNITY 500 spectrometer. The H 2 0 resonance was presaturated. The solution contained 0.8 mM p6 protein in 0.5 M phosphate buffer (pH 5.4)

and 10% DzO. Spectral width in all experiments was 6000 Hz. T e m p e r a t u r e - d e p e n d e n t one-dimensional N M R spectra were measured under the same conditions at 30, 35, 40, 45, 50, 55 and 60°C. In the isotope exchange experiment the 10-fold excess of D 2 0 was added to the solution of the p6 protein in the NMR tube. The first spectrum was taken 2 rain after addition of D20.

Actil,ity of p6 and HIV-I proteinase in t,'itro The biological role of p6 protein was tested in vitro for its possible function as substrate or inhibitor of the HIV-1 proteinase. A 10 -4 M solution of p6 protein in the 0.5 M phosphate buffer of pH 5, 5.4 and 5.7 and containing 1-10 -4 M recombinant HIV-1 proteinase obtained as described earlier [13] was incubated overnight at 40°C. The concentration changes in the sample were monitored as changes in the magnitude of the corresponding peak on the reverse-phase HPLC analysis using water-methanol on the Vydac Cj~ column. Inhibitory activity was tested by use of chromogenic substrate as published by Urban et al. [13] and Richards et al. [14]. In the first experiment 2 . 1 0 -5 M substrate KARVL* FEAM was used with P6 protein concentrations varying from 0 to 10 4 in 0.5 M phosphate buffer pH 5.7. The reaction was started by adding the solution of HIV-1 proteinase to the final concentration of 10-~8 M. The course of the reaction was monitored directly by observation of absorbance change at 305 nm on an Aminco DW-2000 spectrophotometer. In the second experiment 10 -7 M HIV-1 proteinase and p6 protein in concentrations varying from 0 to 10 4 M were pre-incubated in the buffer described above for 2 h. The reaction was started by addition of substrate. The final conditions of the reaction and its measurement were othervise identical to those in previous experiments. Results

The amino acid alignment of p6 protein sequences from different isolates (Fig. 1) shows high conservation of six Pro residues. Up to nine Pro residues were found in some of the isolates. The rest of the sequence is highly hydrophilic. The amino acid composition confers high solubility and flexibility on the protein. In addition, no obvious targets for posttranslational modification were seen in the sequence. The prediction of secondary structure of p6 protein by prediction programs gave contradictory results. Both programs by Chou and Fasman [6,7] predict long, wellstructured regions (Fig. 2a), whereas the program of Garnier et al. [8], on the other hand, predicts only a short six residue segment of long helix (less than two

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Fig. 1. Sequence alignment of different isolates of p6 protein. Sequences for the isolates were taken from the E M B L database of nucleic acid sequences [9]. Highly conserved Pro residues are in bold.

turns) and a negliglible amount of other folded structures (Fig. 2b). The results of the predictions are in disagreement and no consideration can therefore be based on it. Therefore we carried out structure predictions on the sequences from individual isolates, as these do not vary so much from the consensus sequence. The CD spectra of p6 protein (Fig. 3) in the near-ultraviolet region show the content of 100% of 'random' structure according to both spectral databases used for analysis. In addition, the spectrum of p6 is almost identical (within experimental error) to the spectrum presented by Bolotina et al. [12] as random coil spectrum in the protein. The spectrum was not altered by any of the refolding experiments. The one-dimensional NMR spectrum in aqueous solution (Fig. 4) does not show any peaks shifted significantly from their positions found in the random coil structures [15]. The N H protons form several clusters of peaks which are difficult to resolve. In the temperature-dependent spectra all peaks of backbone NH protons are uniformly decreased in intensity and shift simultaneously. This is a clear evidence that all the N H protons are equally exposed to the solution and none of them is buried inside any type of conformation or forms a hydrogen bond to any other atom. The isotope exchange of N H protons to deuterium atoms was too fast to be observed in the time scale of minutes. In the first measurement (2 min) all the NH protons were exchanged with deuterium atoms. It is concluded that no region of the p6 protein has any compact structure and all the N H protons are highly exposed to the solution. The structure is probably extended and allows high exposure of all N H protons to the solution. The sequence - E L Y * P L T (position 36-41 in our alignment of p6 sequences) is similar to those cleaved by the retroviral proteinase. Several more potentially cleavable sequences are present but not utilised by HIV-1 proteinase in the gag and gag-pol encoded

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proteins. In order to ascertain whether the lack of processing of the gag precursor in this site is due to sequence specificity of proteinase or inaccessibility of the site to proteinase, we tested cleavage of p6 protein by HIV-1 proteinase. In the tests of cleavage of p6 no indication was found that the p6 is cleaved by HIV-1 proteinase. We therefore synthesised the 13 and 8 amino acids long peptide fragments PIDKELYPLT0.0 [ 8 ] . 10 - 4 -2.5

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SLR and KELYPLTS to see whether the sequence is cleaved in the form of short flexible peptides. These cleavage experiments were also negative. Clearly, in the particular case of p6 protein no additional cleavage site exists that would fulfill the complex sequential requirement of HIV-1 proteinase. In other experiments we tested the effect of p6 on the cleavage of substrates by HIV-1 proteinase. No inhibition or activation effect of the p6 protein on the rate of cleavage of synthetic substrate was observed in any of the experimental set-ups described above. The p6 protein as such does not affect the activity of HIV-1 proteinase in vitro. We conclude that p6 protein from HIV-1 gag has no compact structure in solution and does not function as the HIV-1 proteinase inhibitor or substrate.

Discussion According to Zybarth et al. [5] gag polyprotein containing p6 is processed in vitro at a significantly lower rate than p6-deficient gag. In this case, provided the inhibitory effect of p6 was excluded, one would expect that a tightly-bound complex is formed between the gag polyprotein and the proteinase. This complex is then slowly cleaved at the N C / p 6 cleavage position and released p6 protein does not further interact with the proteinase. The free proteinase then cleaves the rest of the polyprotein. This complex may play a role in transport of both the polyproteins and the released proteinase to the target location at the cell surface where the final processing may slowly continue. The role of p6 as the core-envelope link [3] in the

late stage of budding is not supported by our observation since one would expect a structured and at least partially hydrophobic protein to interact with the membrane and its components. This role of p6 also does not explain the observation of Gottlinger et al. [2] that the virions in whose genoms the p6 protein-encoding part is shortened instead were partially removed from the viral surface. The number of released virions was greater when the remaining piece of p6 was prolonged. It is, on the other hand, easy to imagine that the longer the remaining piece of p6, the more similar is the final complex to the native one and the greater is then its activity. Other, as yet unknown, mechanisms of p6 function can not be excluded. Structure may be introduced into p6 after its interaction with the target protein in the mature virion, but it is widely accepted that the retroviral proteins adopt their structures in conditions corresponding to those that we have used. We think that attempts to induce structure in p6 by extreme solvent conditions would be difficult and would not resolve the problem that the p6 does not itself adopt a structured conformation.

Acknowledgements The authors thank to Prof. Kurt Wfitrich for help and suggestions. We also thank Dr. Milan Fabry and Prof. John F. Allen for carefully reading the manuscript and for useful comments. The work was supported by Schweizerisches Nationalfonds Grant No. 70TP-029762 and by the grant of the Czechoslovak Academy of Science No. 45501.

161 References I Gottlinger, H.G., Sodroski, J.S. and Haseltine, W.A. (1990) VIII. International Congress of Virology, Berlin, August 1990 (Abstract 15-033). 2 Gottlinger, H.G., Dorfmam T., Sodroski, J.G. and Haseltine, W.A. (1991) Proc. Natl. Acad. Sci., USA. 88, 3195-3199. 3 Gelderblom, H.R. (1991) AIDS, 5, 617-638. 4 Roberts, M.M, and Oroszlan, S. (1989) Biochem. Biophys. Res. Commun. 160, 486-494. 5 Zybarth, G., Partin, K., Ehrich, LS., Wimmer, E., Carter, C.A., Frontiers in HIV therapy, San Diego, 1991 (Abstract 9). 6 Chou, P.Y. and Fasman, G.D. (1978) Adv. Enzymol., 47, 45-148. 7 Chou, P.Y. and Fasman, G.D. (1979) Annu. Rev. Biochem. 47, 251-276.

8 Gamier, J., Osguthorpe, D.J. and Robson, B. (1978), J. Mol. Biol. 120, 97-120.

9 EMBL Data Library, Heidelberg, FRG. 10 Barany, G. and Merrifield, R.B. (1980) The Peptides (Gross, E. and Meyerhofer, J., eds.), Vol. 2, p. 1, Academic Press, New York. 11 Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 13, 3350-3357. 12 Bolotina, I.A., Chekhov, V.O. and Lugauskas, V.Y. (1989) Int. J. Quantum. Chem. 16, 819-825. 13 Urban, J., Konvalinka, J., Stehlikova, J., Gregorova, E., Majer, P., Andreansky, M., Fabry, M., Soucek, P. and Strop, P. (1992) FEBS Lett. 298, 9-13. 14 Richards, A.D., Phylip, L.H., Farmerie, W.G., Scarborough, P.E., Alvarez, A., Dunn, B.M., Hirel, P-H., Konvalinka, J., Strop, P., Pavlickova, L., Kostka, V. and Kay, J. (1990), J. Biol. Chem. 265, 7733-7736. 15 Bundi, A. and Wiitrich, K. (1979) Biopolymers 18, 295-298.